Normal hearing and balance critically depend on the function of mechanosensitive receptors. In the inner ear, these receptors are involved in at least two fundamental processes, transduction by the hair cell stereocilia and regulation of the osmotic pressure of endolymphatic fluid in the scala media. We have recently identified an ion channel that is activated by exposure to osmotic stress and direct mechanical stimulation. This novel receptor, vanilloid receptor-related osmotically activated channel (VR-OAC), is expressed by inner ear hair cells and marginal cells of the stria vascularis. The Vroac gene is a positional candidate for the human deafness disorder DFNA25 and the deaf murine mutant Bronx Waltzer. We hypothesize that it is involved in mechano-reception by hair cells, measurement of the osmotic pressure of endolymphatic fluid, or both. Our long-term goal is to understand the physiological role of VR- OAC and its associated proteins in inner ear function. We will address this question through the parallel accomplishment of four specific aims. We have preliminary evidence that a second mechanoreceptive ion channel, related to VR-OAC, is expressed in the inner ear. We plan to search for additional VR-OAC-like channels (Aim 1). In Aim 2, we will determine the cellular and subcellular localization of VR-OAC and any other related channel that we identify in the inner ear via established methods of in situ hybridization and immunocytochemistry. Because our previous results implicate an essential interaction of VR-OAC with other intracellular proteins, we plan to identify these proteins utilizing the yeast two-hybrid screening system (Aim 3), an approach we have used previously to identify interaction partners of inner ear proteins. In Aim 4, we address the physiological relevance of heteromeric channels formed between VR-OAC and related channels, including the one we already have found. VR- OAC is currently the only mechanoreceptive ion channel that can be assessed by expression in a heterologous system, and we plan to physiologically characterize heteromultimeric channels using calcium imaging and whole cell recordings. We will also test VR- OAC's in vivo role by creating a mouse line carrying a null mutation. Phenotypic assessment of the resulting animals will provide fundamental clues as to the in vivo relevance of VR-OAC for inner ear mechanosensation, or osmoregulation, or both.